Reduction Of IK Research Articles

Human-induced pluripotent stem cell-derived cardiomyocytes to study PUFA analogues as a potential treatment of long QT syndrome.

Long QT syndrome (LQTS) is an inheritable ventricular arrhythmia that predisposes patients to torsades de pointes and sudden cardiac death. Mutations in voltage-gated ion channels (VGC) and their regulatory β subunits involved in the cardiac action potential (AP) generate LQTS by prolonging the QT interval of the electrocardiogram. 15 types of LQTS has been described depending on the underlying gene mutated. LQT1 and LQT5 are described as loss-of-function mutations in genes that encode Kv7.1 (alpha pore-forming subunit) and its β-regulatory subunit KCNE1, respectively. Kv7.1/KCNE1 channels elicit one of the main repolarization currents (IKs) involved in the cardiac AP. Therefore, Kv7.1/KCNE1 channel-specific activators might be a potential treatment for LQTS. Polyunsaturated fatty acid (PUFA) analogues can broadly modulate cardiac VGC when applied on isolated channels in a heterologous system. In the present study, we measured the effects of these four compounds, Linoleoyl-taurine (LIN-Tau), docosahexaenoyl-Tau (DHA-Tau), Linoleoyl-Glycine (LIN-Gly), and DHA-Gly, on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). DHA-analogues shortened the hiPSC-CMs AP duration (APD), while LIN-analogues prolonged hiPSC-CMs APD. DHA-analogues were also tested in isogenic hiPSC-CMs lines for two KCNQ1 (Kv7.1 gene) mutations that cause LQT1, R190Q, and G269S. R190Q is an autosomal dominant missense mutation characterized by a 70-80 % reduction of IKs because of a dominant negative traffic defect. Moreover, we also analyzed DHA-analogues in a heterozygous missense KCNQ1 mutation, G269S. G269S mutation shows a net effect of less than 50% reduction of IKs current (haploinsufficiency). We here use a combination of hiPSC-CM monolayers with a high-resolution, all-optical electrophysiology system for screening the anti-arrhythmic properties of diverse PUFA analogues. Moreover, the well-characterized hiPSC-CMs mutants show us whether PUFA analogues might be a potential treatment approach for more than one type of LQT1.

Docosahexaenoic acid normalizes QT interval in long QT type 2 transgenic rabbit models in a genotype-specific fashion.

Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current. We investigated the effects of DHA in wild type (WT) and transgenic long QT Type 1 (LQT1; loss of IKs), LQT2 (loss of IKr), LQT5 (reduction of IKs), and LQT2-5 (loss of IKr and reduction of IKs) rabbits. In vivo ECGs were recorded at baseline and after 10 µM/kg DHA to assess changes in heart-rate corrected QT (QTc) and short-term variability of QT (STVQT). Ex vivo monophasic action potentials were recorded in Langendorff-perfused rabbit hearts, and action potential duration (APD75) and triangulation were assessed. Docosahexaenoic acid significantly shortened QTc in vivo only in WT and LQT2 rabbits, in which both α- and β-subunits of IKs-conducting channels are functionally intact. In LQT2, this led to a normalization of QTc and of its short-term variability. Docosahexaenoic acid had no effect on QTc in LQT1, LQT5, and LQT2-5. Similarly, ex vivo, DHA shortened APD75 in WT and normalized it in LQT2, and additionally decreased AP triangulation in LQT2. Docosahexaenoic acid exerts a genotype-specific beneficial shortening/normalizing effect on QTc and APD75 and reduces pro-arrhythmia markers STVQT and AP triangulation through activation of IKs in LQT2 rabbits but has no effects if either α- or β-subunits to IKs are functionally impaired. Docosahexaenoic acid could represent a new genotype-specific therapy in LQT2.

Exaggerated potassium current reduction by oxytocin in visceral sensory neurons following chronic intermittent hypoxia

Oxytocin (OT) from the hypothalamus is increased in several cardiorespiratory nuclei and systemically in response to a variety of stimuli and stressors, including hypoxia. Within the nucleus tractus solitarii (nTS), the first integration site for cardiorespiratory reflexes, OT enhances synaptic transmission, action potential (AP) discharge, and cardiac baroreflex gain. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. The nTS receives sensory input from baroafferent neurons that originate in the nodose ganglia. Nodose neurons express the OT receptor (OTR) whose activation elevates intracellular calcium. However, the influence of OT on other ion channels, especially potassium channels important for neuronal activity during CIH, is less known. This study sought to determine the mechanism (s) by which OT modulates sensory afferent-nTS mediated reflexes normally and after CIH. Nodose ganglia neurons from male Sprague-Dawley rats were examined after 10d CIH (6% O2 every 3min) or their normoxic (21% O2) control. OTR mRNA and protein were identified in Norm and CIH ganglia and was similar between groups. To examine OTR function, APs and potassium currents (IK) were recorded in dissociated neurons. Compared to Norm, after CIH OT depolarized neurons and reduced current-induced AP discharge. After CIH OT also produced a greater reduction in IK that where tetraethylammonium-sensitive. These data demonstrate after CIH OT alters ionic currents in nodose ganglia cells to likely influence cardiorespiratory reflexes and overall function.

High Efficacy by GAL-021: A Known Intravenous Peripheral Chemoreceptor Modulator that Suppresses BKCa-Channel Activity and Inhibits IK(M) or Ih.

GAL-021 has recently been developed as a novel breathing control modulator. However, modifications of ionic currents produced by this agent remain uncertain, although its efficacy in suppressing the activity of big-conductance Ca2+-activated K+ (BKCa) channels has been reported. In pituitary tumor (GH3) cells, we found that the presence of GAL-021 decreased the amplitude of macroscopic Ca2+-activated K+ current (IK(Ca)) in a concentration-dependent manner with an effective IC50 of 2.33 μM. GAL-021-mediated reduction of IK(Ca) was reversed by subsequent application of verteporfin or ionomycin; however, it was not by that of diazoxide. In inside-out current recordings, the addition of GAL-021 to the bath markedly decreased the open-state probability of BKCa channels. This agent also resulted in a rightward shift in voltage dependence of the activation curve of BKCa channels; however, neither the gating charge of the curve nor single-channel conductance of the channel was changed. There was an evident lengthening of the mean closed time of BKCa channels in the presence of GAL-021, with no change in mean open time. The GAL-021 addition also suppressed M-type K+ current with an effective IC50 of 3.75 μM; however, its presence did not alter the amplitude of erg-mediated K+ current, or mildly suppressed delayed-rectifier K+ current. GAL-021 at a concentration of 30 μM could also suppress hyperpolarization-activated cationic current. In HEK293T cells expressing α-hSlo, the addition of GAL-021 was also able to suppress the BKCa-channel open probabilities, and GAL-021-mediated suppression of BKCa-channel activity was attenuated by further addition of BMS-191011. Collectively, the GAL-021 effects presented herein do not exclusively act on BKCa channels and these modifications on ionic currents exert significant influence on the functional activities of electrically excitable cells occurring in vivo.

Role of infarct scar dimensions, border zone repolarization properties and anisotropy in the origin and maintenance of cardiac reentry

Cardiac ventricular tachycardia (VT) is a life-threatening arrhythmia consisting of a well organized structure of reentrant electrical excitation pathways. Understanding the generation and maintenance of the reentrant mechanisms, which lead to the onset of VT induced by premature beats in presence of infarct scar, is one of the most important issues in current electrocardiology. We investigate, by means of numerical simulations, the role of infarct scar dimension, repolarization properties and anisotropic fiber structure of scar tissue border zone (BZ) in the genesis of VT. The simulations are based on the Bidomain model, a reaction-diffusion system of Partial Differential Equations, discretized by finite elements in space and implicit-explicit finite differences in time. The computational domain adopted is an idealized left ventricle affected by an infarct scar extending transmurally. We consider two different scenarios: i) the scar region extends along the entire transmural wall thickness, from endocardium to epicardium, with the exception of a BZ region shaped as a central sub-epicardial channel (CBZ); ii) the scar region extends transmurally along the ventricular wall, from endocardium to a sub-epicardial surface, and is surrounded by a BZ region (EBZ). In CBZ simulations, the results have shown that: i) the scar extent is a crucial element for the genesis of reentry; ii) the repolarization properties of the CBZ, in particular the reduction of IKs and IKr currents, play an important role in the genesis of reentrant VT. In EBZ simulations, since the possible reentrant pathway is not assigned a-priori, we investigate in depth where the entry and exit sites of the cycle of reentry are located and how the functional channel of reentry develops. The results have shown that: i) the interplay between the epicardial anisotropic fiber structure and the EBZ shape strongly affects the propensity that an endocardial premature stimulus generates a cycle of reentry; ii) reentrant pathways always develop along the epicardial fiber direction; iii) very thin EBZs rather than thick EBZs facilitate the onset of cycles of reentry; iv) the sustainability of cycles of reentry depends on the endocardial stimulation site and on the interplay between the epicardial breakthrough site, local fiber direction and BZ rim.

Oxytocin Reduction of Nodose Ganglion Neurons Potassium Currents is Enhanced in Chronic Intermittent Hypoxia (CIH)

Oxytocin (OT) from the hypothalamus is increased in several cardiorespiratory nuclei and the circulation in response to a variety of stimuli and stressors, including hypoxia. Oxytocin within the nucleus tractus solitarii (nTS) enhances synaptic transmission, action potential (AP) discharge, and cardiac baroreflex gain to lower heart rate. The baroreceptor reflex is mediated through afferent neurons whose somas originate in the nodose ganglia and project centrally to the nTS. Nodose neurons express the OT receptor, and its activation elevates intracellular calcium; the influence of OT on other ion channels, especially during hypoxia, is not known. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. Recent studies suggest enhancing OT in the dorsal vagal complex during CIH may reduce blood pressure. This study was undertaken to determine the mechanism(s) by which OT modulates sensory afferent‐nTS mediated reflexes normally and after CIH. Nodose neurons of 5–6 wk old male Sprague‐Dawley rats were isolated after 10 d of Normoxia (21% O2, Norm) or CIH (alternating 21% & 6% O2, 8 hr/day). Two‐5 hrs after isolation, we recorded outward potassium currents (IK) and APs by the patch clamp method. To examine IK, neurons were held at −60 mV and currents were evoked by depolarizing voltages (−70 to +60 mV, 140 ms). The steady state IK‐voltage relationship was analyzed. AP discharge was evoked by injecting 50 pA depolarizing steps (−20 to 280 pA, 200 ms); the current‐discharge relationship was evaluated. IK and APs were examined during baseline and after 3 min of vehicle or OT (300, 600, or 1000 nM). Vehicle did not alter IK in Norm (n=6) and CIH (n=4) cells, confirming lack of time‐dependent IK changes. In Norm cells, OT decreased IK at voltages greater than +40 mV (300 nM, n=8) and +60 mV (600 nM, n=7). After CIH, OT (300 nM, n=5) also decreased IK at +40 mV and greater. OT at 600 nM (n=5) attenuated IK at significantly lower voltages (+20 mV) in CIH vs. Norm. OT‐sensitive currents at +60 mV were greater in CIH than Norm at 300 and 600 nM. In Norm and CIH groups (n=4, 5), OT at 1000 nM had variable IK responses which overall were not significantly different from baseline. Fitting IKs to Boltzmann functions demonstrated a reduction in half‐activation during OT in Norm cells. In Norm and CIH cells, application of the K+ channel blocker tetraethylammonium (TEA, 10 mM) decreased IK and blocked the OT‐induced reduction in IK (600 nM, n=3). Depolarizing nodose neurons with up to 280 pA injection induced APs in Norm (n=7) and CIH (n=5) neurons, and 600 nM OT did not alter the number of APs during a given current step. Yet, 600 nM OT decreased AP threshold and tended to increase AP half‐width in CIH but not Norm cells, consistent with the reduction in IK by OT in CIH. In Norm cells, vehicle and OT did not alter holding currents measured at −60 mV, but after CIH 600 nM OT induced modest depolarizing inward currents. Taken together, these data demonstrate OT induces changes in ionic currents in nodose ganglia cells that are consistent with increasing excitability in CIH, and may also shed light on the bradycardic and anti‐hypertensive action of OT.Support or Funding InformationRO1 HL 128454 & HL 098602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GW24-e0353 A novel mutation R397Q in KCNQ1 gene caused dilated cardiomyopathy with ventricular tachycardia and prolonged QT interval

ObjectivesDilated cardiomyopathy (DCM) is a major cause of sudden cardiac death with lethal ventricular arrhythmias. So far, the genetic mutations of DCM are mainly related to the sarcomeric protein, and...

Modeling [Ca2+]o- and [K+]o-dependent oscillations in spinal Hb9 interneurons

The spinal interneurons in newborn rodents, when synaptically isolated by removing the extracellular calcium ([Ca2+]o), demonstrate intrinsic rhythmic bursting activity that can be suppressed by riluzole, a blocker of the persistent sodium current (INaP) [2]. This finding led to the suggestion that lowering of [Ca2+]o may enhance INaP by shifting its activation threshold toward more negative voltages, and raised the question of functional relevance of this finding to generation of locomotor rhythm. To assess this issue, a series of experiments was performed in vitro using the isolated spinal cord preparation from the neonatal rat with measurements of [Ca2+]o and extracellular potassium concentration ([K+]o) during pharmacologically induced fictive locomotion. We demonstrated that with the onset of fictive locomotion, [Ca2+]o reduced from 1.2 up to 0.9 mM whereas [K+]o increased from 4 up to 6 mM. At the same time, a special study performed on the isolated genetically identified Hb9 excitatory interneurons showed that, at [Ca2+]o= 1 mM and [K+]o=5 mM, 12% of Hb9 cells expressed intrinsic INaP-dependent bursting, and at the concentrations typical for fictive locomotion ([Ca2+]o= 0.9 mM and [K+]o=6 mM), as many as 50% of identified Hb9 interneurons expressed INaP-dependent bursting. Importantly, the threshold of [Ca2+]o to generate bursting decreased as [K+]o increased. The analysis of Hb9 neuron behavior during slow ramp increase of voltage revealed that lowering [Ca2+]o from 1.2 to 0.9 mM induced a negative shift (~ -3 mV) in the INaP half-activation voltage (V1/2NaP). In contrast, V1/2NaP was not changed when [K+]o increased from 4 to 6 mM. To theoretically investigate the effect of changing [Ca2+]o and [K+]o on the Hb9’s pacemaker properties and firing behavior, we developed a single-compartment computational model of Hb9 neuron. In this model, we explicitly simulated a negative shift of V1/2NaP occurring with the reduction of [Ca2+]o.. At [K+]o=6 mM, our model exhibited tonic activity at V1/2NaP = –50 mV (Fig. ​(Fig.1A,1A, left). The rhythmic bursting emerged at V1/2NaP = –51 mV, and further shifting V1/2NaP to the left produced stable bursting (Fig. ​(Fig.1A,1A, right). In turn, an increase in [K+]o reduced the potassium reversal potential and hence all voltage-gated potassium currents (IK), which provided an additional augmentation of INaP-dependent bursting [1]. To study a synergistic effect of [Ca2+]o and [K+]oon the emergence of bursting activity, we modeled a population of 50 uncoupled neurons with randomly distributed parameters (see Fig. ​Fig.1B).1B). Our simulations have shown that shifting V1/2NaP towards more negative values induced by reducing [Ca2+]o may play a major role in emergence of bursting activity in the population of spinal interneurons. We have also demonstrated that accumulation of [K+]ocan facilitate the emergence of INaP-dependent bursting via the reduction of IK. Figure 1 A. Switching from tonic to bursting activity in the modeled neuron by shifting V1/2NaP to more negative value. B. The synergistic effect of [Ca2+]o and [K+]ochanges on emergence of bursting activity in the modeled population of 50 uncoupled cells. In summary we suggest that co-regulation of INaP and IK by the corresponding changes in [Ca2+]o and [K+]o may convert activity of spinal interneurons from asynchronous/tonic to the synchronized bursting. This activity-dependent switching in firing behavior may represent a fundamental mechanism for locomotor rhythm generation in the spinal cord.

Regulation of the instantaneous inward rectifier and the delayed outward rectifier potassium channels by Captopril and Angiotensin II via the Phosphoinositide-3 kinase pathway in volume-overload-induced hypertrophied cardiac myocytes

SummaryBackgroundEarly development of cardiac hypertrophy may be beneficial but sustained hypertrophic activation leads to myocardial dysfunction. Regulation of the repolarizing currents can be modulated by the activation of humoral factors, such as angiotensin II (ANG II) through protein kinases. The aim of this work is to assess the regulation of IK and IK1 by ANG II through the PI3-K pathway in hypertrophied ventricular myocytes.Material/MethodsCardiac eccentric hypertrophy was induced through volume-overload in adult male rats by aorto-caval shunt (3 weeks). After one week half of the rats were given captopril (2 weeks; 0.5 g/l/day) and the other half served as control. The voltage-clamp and western blot techniques were used to measure the delayed outward rectifier potassium current (IK) and the instantaneous inward rectifier potassium current (IK1) and Akt activity, respectively.ResultsHypertrophied cardiomyocytes showed reduction in IK and IK1. Treatment with captopril alleviated this difference seen between sham and shunt cardiomyocytes. Acute administration of ANG II (10−6M) to cardiocytes treated with captopril reduced IK and IK1 in shunts, but not in sham. Captopril treatment reversed ANG II effects on IK and IK1 in a PI3-K-independent manner. However in the absence of angiotensin converting enzyme inhibition, ANG II increased both IK and IK1 in a PI3-K-dependent manner in hypertrophied cardiomyocytes.ConclusionsThus, captopril treatment reveals a negative effect of ANG II on IK and IK1, which is PI3-K independent, whereas in the absence of angiotensin converting enzyme inhibition IK and IK1 regulation is dependent upon PI3-K.

Modification of activation kinetics of delayed rectifier K+ currents and neuronal excitability by methyl-β-cyclodextrin

The effects of methyl-β-cyclodextrin (MβCD), an oligosaccharide, on ion currents were investigated in differentiated NG108-15 neuronal cells. In NG108-15 cells treated with dibutyryl cyclic AMP, the expression level of the KV3.1 mRNA was elevated. Depletion of membrane cholesterol by exposing cells to MβCD (1 mM) resulted in a significant reduction of the activation kinetics of delayed rectifier K+ current (IK(DR)) in these cells. However, neither activation nor inactivation curve of IK(DR) was altered following MβCD treatment. In current-clamp recordings, in MβCD-treated cells, the instantaneous frequency of the firing in response to long-lasting current stimuli was reduced. In a modified Hodgkin-Huxley neuron, the upward shift in the relationship of activation/deactivation time constant of IK(DR) versus membrane potential causes a reduction of IK(DR) amplitude accompanied by an increase in the width of action potentials. In the studies from a high-frequency modeled neuron, reduction of voltage-dependent activation of IK(DR) can also facilitate spike-frequency adaptation. In a simulated network of spiking neurons, the increased activation/deactivation time constant of IK(DR) slowed repetitive firing. Taken together, MβCD may slow activation kinetics of IK(DR) and confer a trigger for the propensity to develop spike-frequency adaptation in neurons or neuroendocrine cells.

Rb+ Efflux Through Functional Activation of Cardiac KCNQ1/minK Channels by the Benzodiazepine R-L3 (L-364,373)

The slow delayed rectifier K+ current, Iks, encoded by KCNQ1 (KvLQT1)/KCNE1 (mink) genes, contributes to cardiac action potential repolarization and determines the heartbeat rate. Mutations in either KCNQ1 or KCNE1 that reduce Iks cause long-QT syndrome (LQTS), a disorder of ventricular repolarization that results in cardiac arrhythmia and sudden death. A well-recognized potential treatment for LQTS caused by reduction of Iks is to enhance functional activation of cardiac KCNQ1/KCNE1 channels. In the present study, we generated a stable Chinese hamster ovary cell line that expresses KCNQ1/KCNE1 channels confirmed by electrophysiology. Using a pharmacological tool compound R-L3 (L-364,373 [(3-R)-1,3-dihydro-5-(2-fluorophenyl)-3-(1H-indol- 3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one]), which activates KCNQ1/mink channels, we then developed and validated a non-radioactive rubidium (Rb+) efflux assay that directly measures the functional activity of KCNQ1/KCNE1 channels by atomic absorption spectroscopy. Our results show that the validated Rb+ efflux assay can be used for screening of KCNQ1/KCNE1 openers that potentially treat LQTS in both inherited and acquired forms. In addition, the assay also can be used for evaluation of possible long-QT liability during cardiac selectivity of new chemical entities.

Melatonin inhibits outward delayed rectifier potassium currents in hippocampal CA1 pyramidal neuron via intracellular indole-related domains.

In the present study, we investigated the effect of melatonin on the outward delayed rectifier potassium currents (IK) in CA1 pyramidal neurons of rat hippocampal slices using patch-clamp technique in whole-cell configuration. In a concentration-dependent manner, melatonin caused a reduction of IK with a half-maximal inhibitory concentration (IC50) of 3.75 mm. The inhibitory effect had rapid onset and was readily reversible. Melatonin shifted steady-state inactivation of IK in hyperpolarizing direction but did not alter its steady-state activation. Neither luzindole, an MT1/MT2 receptor antagonist, nor prazosin, an MT3 receptor antagonist, blocked melatonin-induced current reduction. The results indicate that melatonin-induced IK inhibition was not via activation of its own membrane receptors. 5-Hydroxytryptamine (5-HT), a melatonin precursor and an agonist of serotonin receptors, when it was given in pipette internal solution but not bath solution, produced a similar inhibitory effect to that of melatonin. Moreover, indole, a major component of melatonin, reversibly and dose dependently inhibited IK with an IC50 of 3.44 mm. Present results suggest that melatonin inhibits IK in hippocampal CA1 pyramidal neurons probably through its interaction with the intracellular indole-related domains of potassium channels.

Mutations Affecting the Delayed Rectifier Potassium Current in Drosophila

Mutations and pharmacological agents have been used to resolve and analyze several K+ currents in Drosophila. Mutations that affect channels carrying the voltage-activated IA and the Ca2+-activated ICF have helped greatly in analyzing the structure, function and regulation of these channels. We now report mutations that selectively affect the delayed rectifier current, IR. Flies mutagenized with ethylmethanesulfonate were screened for temperature-induced paralysis. Paralytic mutants identified in the screen were examined for K+ currents in the larval body-wall muscles. The z66 mutant larvae showed a significant reduction in IK. The mutation did not affect other K+ currents (IA, ICF, or ICS) or the Ca2+ channel current in the muscles. Another mutation, z4, which showed reduced IK, failed to complement z66. Genetic analysis localized the gene disrupted by z66 and z4 to the left arm of chromosome 3, in the 63A1–63B6 region on polytene chromosomes. The z66 and the z4 mutations, which lie in the Shab K+ channel gene, provide an opportunity to undertake analysis of the functioning of these channels and to study the role of these channels in membrane excitability.

Swelling‐induced decrease in spontaneous pacemaker activity of rabbit isolated sino‐atrial node cells

The heart responds to an increase in sino-atrial node wall stress with an augmentation in rate of contraction. It has been suggested that swelling-activated ion channels may play a key role in that response. This paper investigates directly the effects of cell swelling on spontaneous activity of rabbit isolated sino-atrial node pacemaker cells. The main finding is that sino-atrial node cells, studied in current clamp mode using amphotericin-permeabilized patches, decrease their spontaneous pacemaker rate by 24.2 +/- 7.8% (P < 0.01, n = 9) during 75% hyposmotic swelling. This response is opposite to the predicted impact of volume-activation of sarcolemmal ion conductances. Computer modelling (OXSOFT Heart v4.8) suggests that swelling-induced dilution of the cytosol, reduction in intracellular potassium concentration, and decrease in the delayed rectifier potassium current, IK, are leading mechanisms in the response. This is supported by voltage-clamp data that show a swelling-induced positive shift in the reversal potential of IK by between 5 and 10 mV (n = 7) and a reduction in amplitude of its rapidly activating component, IKr, (n = 6). Thus, spontaneously active sino-atrial node cells reduce pacemaking rate during swelling. This response cannot be explained by the known volume-activated sarcolemmal ion conductances, but appears to be dictated by other mechanisms including dilution of the cytosol and reduction in IK. The results re-enforce the view that cardiac responses to cell volume changes may be quite different from those to longitudinal stretch.

Isoflurane Reduces K+ Current in Single Smooth Muscle Cells of Guinea Pig Portal Vein

Volatile anesthetics vasodilate in part by direct action on vascular smooth muscle.Isoflurane-induced relaxation of portal vein smooth muscle involves alteration of membrane ionic currents that control cell excitability and contraction. Whole cell voltage clamp technique was used to examine outward Ca2+-activated K+ current (IK,Ca) in guinea pig portal vein cells. Isoflurane caused a concentration-dependent reduction in IK,Ca at steady-state conditions but had no significant effect on resting potential. Isoflurane transiently potentiated IK,Ca by a mechanism that may partly involve Ca2+ release from intracellular storage sites. The depression of IK,Ca by isoflurane may occur by direct action on the channel protein or on the lipid environment of the channel to alter conductance or kinetic properties. Since isoflurane reduces IK,Ca coincident with suppression of Ca2+ channel current, it was concluded that the depression of IK,Ca by isoflurane is of secondary importance to reduction in inward Ca2+ channel current. Potentiation of IK,Ca may preclude significant membrane activation during the onset of isoflurane's action. (Anesth Analg 1996;83:1307-13)

Isoflurane reduces K+ current in single smooth muscle cells of guinea pig portal vein.

Volatile anesthetics vasodilate in part by direct action on vascular smooth muscle. Isoflurane-induced relaxation of portal vein smooth muscle involves alteration of membrane ionic currents that control cell excitability and contraction. Whole cell voltage clamp technique was used to examine outward Ca(2+)-activated K+ current (IK,Ca) in guinea pig portal vein cells. Isoflurane caused a concentration-dependent reduction in IK,Ca at steady-state conditions but had no significant effect on resting potential. Isoflurane transiently potentiated IK,Ca by a mechanism that may partly involve Ca2+ release from intracellular storage sites. The depression of IK,Ca by isoflurane may occur by direct action on the channel protein or on the lipid environment of the channel to alter conductance or kinetic properties. Since isoflurane reduces IK,Ca coincident with suppression of Ca2+ channel current, it was concluded that the depression of IK,Ca by isoflurane is of secondary importance to reduction in inward Ca2+ channel current. Potentiation of IK,Ca may preclude significant membrane activation during the onset of isoflurane's action.

Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms.

Angiotensin II (ANG II) elicits an ANG II type 1 (AT1) receptor-mediated decrease in voltage-dependent K+ current (Ik) and an increase in voltage-dependent Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem. Modulation of these currents by ANG II involves intracellular messengers that result from an AT1 receptor-mediated stimulation of phosphoinositide hydrolysis. For example, the effects of ANG II on IK and ICa were abolished by phospholipase C antagonists. The reduction in IK produced by ANG II was attenuated by either protein kinase C (PKC) antagonists or by chelation of intracellular Ca2+. By contrast, PKC antagonism abolished the stimulatory effect of ANG II on ICa. Superfusion of the PKC activator phorbol 12-myristate 13-acetate produced effects on IK and ICa similar to those observed after ANG II. Furthermore, intracellular application of inositol 1,4,5-trisphosphate (IP3) elicited a significant reduction in IK. This suggests that the AT1 receptor-mediated changes in neuronal K+ and Ca2+ currents involve PKC (both IK and ICa) and IP3 and/or intracellular Ca2+ (IK).

Functional interaction between K(ATP) channels and the Na(+)-K(+) pump in metabolically inhibited heart cells of the guinea-pig.

1. Transmembrane current through ATP-regulated K(+) channels (IK(ATP)) was measured in ventricular heart cells of the guinea-pig in the whole-cell and cell-attached patch configurations under conditions of metabolic poisoning with the mitochondrial uncoupler 2,4-dinitrophenol (DNP). 2. Maintained exposure of the cells to DNP resulted in a transient appearance of whole-cell IK(ATP) When IK(ATP) had reached several nanoamps, blocking the forward-running Na(+)-K(+) pump with 0.5 mM strophanthidin decreased IK(ATP) after a delay. The time course of this decrease could be described by a single exponential function, which yielded a time constant(T)of 4.51+/- 1.89 s (n=8). 3. Hyperpolarization from 0 mV to -100 or -150 mV for 2 s caused IK(ATP) (measured at 0 mV) to decrease by 34.2 +/- 14.1 % (n = 8) and 37.6 +/- 9.4% (n = 8), respectively. After the hyperpolarizing pulse, IK(ATP) returned to its higher initial level within a couple of seconds. 4. Driving the pump backwards by removing the extracellular K(+) ions caused the permanent disappearance of DNP-induced IK(ATP). 5. Application of 0.5 mM strophanthidin in the absence of external K(+) ions induced a transient increase in IK(ATP), as did washing out the glycoside (n = 5). 6. When pump action was inhibited by using Na(+), K(+)-free Tyrode solution (see Methods) in the bath, strophanthidin did not have a comparable direct effect on IK(ATP). 7. In cell-attached patches, strophanthidin applied via the bath caused a reduction in IK(ATP) with a similar time course to that in whole-cell experiments. This suggests that the interaction between the pump molecules and the K(ATP) channels is not restricted to closely neighbouring molecules. 8. The data support the hypothesis that [ATP] at the cytosolic face of the membrane may drop to practically zero, thereby passing an 'ATP window' in which the channels first open and then close, and that the submembrane [ATP] is readily controlled by the cytosolic [ATP].

Activation of a nonselective cationic conductance by metabotropic glutamatergic and muscarinic agonists in CA3 pyramidal neurons of the rat hippocampus

We have characterized a cationic membrane conductance activated by metabotropic glutamatergic and muscarinic cholinergic agonists in CA3 neurons in hippocampal slice cultures using the patch-clamp technique. When the potassium concentration in the superfusing fluid was raised above 5 mM, a biphasic current was observed in cells held at -60 mV in response to stimulation of postsynaptic metabotropic glutamate receptors (mGluRs) with 1S,3R-ACPD (50 microM) or muscarinic receptors with methacholine (MCh, 5 microM). The initial inward component was due to an increase in a cationic membrane conductance as determined by its reversal potential and its sensitivity to changes in extracellular K+ or Na+. The conductance underlying this current displayed no apparent voltage sensitivity over the range -120 to -50 mV. The response was reduced by extracellular application of Ba2+, Cd2+, Mg2+, or TEA, whereas extracellular Cs+ or loading cells with BAPTA or Cs+ did not affect the current. The effects of 1S,3R-ACPD were reversibly inhibited by bath-applied MCPG, an antagonist at mGluRs. Experiments with atropine and pirenzepine indicated that non-M1 muscarinic receptors mediated the MCh-induced current. A decrease in a resting leak potassium conductance (IK,leak) was responsible for the late component of the 1S,3R-ACPD- and MCh-induced response, seen as an outward current in the bathing solution with high K+ concentration. Loading cells with GDP beta S, GTP gamma S, or GTP did not alter the cationic current, while, in the same cells, the reduction in IKleak was abolished or irreversibly activated. Single-channel recordings of cationic channel activity in the cell-attached configuration provided evidence for the requirement of second messengers in coupling these receptors to the cationic channels. The data indicate that in addition to the previously described reduction of IK,leak, IM, and IAHP, both 1S,3R-ACPD and MCh activate a nonselective cationic conductance that is clearly revealed upon elevating external K+ concentration. This current is mediated by activation of metabotropic receptors, although no evidence could be obtained to show an involvement of G-proteins.

Inhibition of delayed rectifier K(+)-current by levcromakalim in single intestinal smooth muscle cells: effects of cations and dependence on K(+)-flux.

1. Whole-cell voltage-clamp recordings were made from single smooth muscle cells isolated from the longitudinal layer of the guinea-pig small intestine. 2. Levcromakalim ((-)Ckm) inhibited delayed rectifier K-current (IK(DR)) and induced a voltage-independent K-current (IK(-Ckm)). Both effects were inhibited similarly by glibenclamide. In some cells, however, IK(-Ckm) could be induced without any effect on IK(DR). 3. Ba2+ caused a voltage-dependent block of IK(-Ckm). The IC50 was 0.2 mM at -40 mV (6 cells), but at 0 mV 2 mM Ba2+ caused only a 26 +/- 7% inhibition (n = 5). Ba2+ had much less effect on IK(DR), 2 mM Ba2+ having no inhibitory effect on current elicited by depolarization to -30 mV (n = 6) or 0 mV (n = 5). 4. Low concentrations of Zn2+ blocked IK(-Ckm) while having little effect on IK(DR). Zn2+ (40 microM) caused a 77 +/- 1% reduction of IK(-Ckm) at -30 mV (n = 4) but IK(DR) was inhibited by only 10 +/- 3% at the same voltage (n = 4). 5. Inward current amplitudes were compared in 135 mM Rb+ and 135 mM K+ bath solutions. (-)Ckm-activated Rb(+)-current was only 4% of the K(+)-current, whereas delayed rectifier Rb(+)-current was larger than K(+)-current. 6. (-)Ckm did not inhibit IK(DR) if IK(-Ckm) was blocked. In the presence of 2 mM Ba2+ or 135 mM Rb+, (-)Ckm did not induce current nor did it inhibit the delayed rectifier. When [Rb+]o was 25 mM and [K+]J was 130 mM, (-)Ckm elicited outward current and inhibited outward delayed rectifier current (at voltages positive of the reversal potential) but it did not elicit inward current or inhibit inward delayed rectifier current (at voltages negative of the reversal potential).7. These experiments indicate that (-)Ckm-activated K channels are more sensitive to inhibition by Ba2+and Zn2+ and pass inward Rb+ current less well than delayed rectifier K channels. They also suggest that (-)Ckm does not modulate delayed rectifier K channels directly or via an intermediate protein but that the inhibitory effect of (-)Ckm on IK(DR) arises as a consequence of K+-flux through (-)Ckm activated K channels.